Isolation and genetic dissection of an eukaryotic replicon that supports autonomous DNA replication
dc.contributor | Kapler, Geoffrey M. | |
dc.creator | Datta, Shibani | |
dc.date.accessioned | 2007-04-25T20:01:13Z | |
dc.date.accessioned | 2017-04-07T19:52:35Z | |
dc.date.available | 2007-04-25T20:01:13Z | |
dc.date.available | 2017-04-07T19:52:35Z | |
dc.date.created | 2005-12 | |
dc.date.issued | 2007-04-25 | |
dc.description.abstract | Maintenance of genome integrity requires that chromosomes be accurately and faithfully replicated. We are using Tetrahymena thermophila as a model system for studying the initiation and regulation of eukaryotic DNA replication. This organism contains a diploid micronucleus and polyploid macronucleus. During macronuclear development, the five diploid chromosomes of the micronucleus are fragmented into 280 macronuclear minichromosomes that are subsequently replicated to ~45 copies. In stark contrast, the 21 kb ribosomal DNA minichromosome (rDNA) is amplified from 2 to 10,000 copies in the same nucleus. Previous characterization of the rDNA replicon has led to the localization of its origin and the cis-acting regulatory determinants to the 1.9 kb 5'non-transcribed spacer region. The objective of this study was to identify and characterize non-rDNA origins of replication in Tetrahymena. This will help determine the underlying basis for differential regulation of rDNA and non-rDNA origins during development, as well as provide a better understanding of the organization of eukaryotic replicons. To this effect, I developed a DNA transformation assay that I used to isolate new Tetrahymena replication origins. A 6.7 kb non-rDNA fragment, designated TtARS1, was shown to support stable autonomous replication of circular plasmids in Tetrahymena. Genetic dissection revealed that TtARS1 contains two independent replicons, TtARS1-A and TtARS1-B. Full TtARS1-A function requires a minimal sequence of 700 bp, and two small regions in this fragment have been shown to be essential for origin function. TtARS1-B replicon function was localized to a 1.2 kb intergenic segment that contains little sequence similarity to TtARS1-A. Both non-rDNA replicons lack sequence similarity to the rDNA 5' NTS, suggesting that each replicon interact with a different set of regulatory proteins. This study indicates that the rDNA and the non-rDNA replicons have a modular organization, containing discrete, cis-acting replication determinants. | |
dc.identifier.uri | http://hdl.handle.net/1969.1/4666 | |
dc.language.iso | en_US | |
dc.publisher | Texas A&M University | |
dc.subject | eukaryotic | |
dc.subject | DNA replication | |
dc.title | Isolation and genetic dissection of an eukaryotic replicon that supports autonomous DNA replication | |
dc.type | Book | |
dc.type | Thesis |